NOVEL IMMUNOTHERAPIES TARGETING PD-1 WITH ANTI-PD-1/IL-15 IMMUNOCYTOKINES

Abstract

The inventors now provide novel IL-15/IL-15 receptor alpha (IL-15Rα) fusion proteins. Furthermore, as a complement to anti-PD-1 therapy, the inventors developed a series of anti-PD-1/IL-15/IL-15 receptor alpha (IL-15Rα) immunocytokines that are able to simultaneously target multiple steps in the immune activation process. The development of said immunocytokines provides the potential benefits associated with anti-PD-1 antibodies and IL-15 administered individually with several distinct advantages. These include a significantly extended in vivo half-life relative to the IL-15 therapy, administration of a pre-formed IL-15/IL-15Rα complex that would preclude the need for IL-15Rα trans-presentation, high activity leading to a low target therapeutic dose and targeted delivery of IL-15 to regions with high PD-1 cells that will limit off-target adverse events.

Claims

1. An IL-15/IL-15 receptor alpha (IL-15Rα) fusion protein comprising i) a IL15-R alpha sushi-containing polypeptide comprising an amino acid sequence having at least 80% of identity with the amino acid sequence of SEQ ID NO:1 ii) a linker having an amino acid sequence as set forth in SEQ ID NO:2 and iii) an IL-15 polypeptide comprising the amino acid sequence having at least at least 80% of identity with the amino acid sequence of SEQ ID NO:3; a nucleic acid encoding the IL-15/IL-15 receptor alpha fusion protein, a vector that comprises the nucleic acid or a host cell which has been transfected, infected or transformed by the nucleic acid.

2. The IL-15/IL-15 receptor alpha fusion protein of claim 1, wherein the IL-15/IL-15 receptor alpha fusion protein has the amino acid sequence as set forth in SEQ ID NO:4.

3. A heavy chain of an antibody that is fused to the IL-15/IL-15 receptor alpha fusion protein of claim 1, a nucleic acid encoding the heavy chain, a vector that comprises the nucleic acid or a host cell which has been transfected, infected or transformed by the nucleic acid.

4. The heavy chain of claim 3, wherein the heavy chain is fused to the IL-15/IL-15 receptor alpha fusion protein via a linker.

5. The heavy chain of claim 4 wherein the linker comprises the amino acid sequence as set forth in SEQ ID NO:5.

6. The heavy chain of claim 5 wherein the linker has the amino acid sequence as set forth in SEQ ID NO:6.

7. The heavy chain of claim 3, wherein the heavy chain is from an antibody having specificity for PD-1.

8. The heavy chain of claim 7, wherein the heavy chain comprises a VH domain as set forth in SEQ ID NO:7, 8 or 9.

9. The heavy chain of claim 7, wherein the heavy chain comprises an IgG Fc region of an IgG4 immunoglobulin.

10. The heavy chain of claim 7, wherein the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:10, 11 or 12.

11. The heavy chain of claim 7, wherein the heavy chain has the amino acid sequence as set forth in SEQ ID NO:13, 14, or 15.

12. An immunocytokine a heavy chain of an antibody that is fused to the IL-15/IL-15 receptor alpha fusion protein of claim 1, a nucleic acid that encodes the immunocytokine, a vector that comprises the nucleic acid or a host cell which has been transfected, infected or transformed by the nucleic acid.

13. The immunocytokine claim 12, wherein the immunocytokine has specificity for PD-1.

14. The immunocytokine of claim 12 wherein the heavy chain has the amino acid sequence as set forth in SEQ ID NO:13, 14, or 15.

15. The immunocytokine of claim 12, wherein the immunocytokine comprises: a heavy chain a set forth in SEQ ID NO:13 and a light chain a set forth in SEQ ID NO:16, a heavy chain a set forth in SEQ ID NO:14 and a light chain a set forth in SEQ ID NO:17 or a heavy chain a set forth in SEQ ID NO:15 and a light chain a set forth in SEQ ID NO:18.

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. The nucleic acid of claim 3, wherein the nucleic acid comprises the nucleic acid sequence as set forth in SEQ ID NO:19 or 20.

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. A method of reducing T cell exhaustion, treating cancer or treating an infectious disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one anti-PD-1 immunocytokine of claim 13.

28. (canceled)

29. (canceled)

30. The method of claim 27, wherein the infectious disease is a viral infection caused by a single or double stranded RNA or a DNA virus, which infects animals, humans and plants, wherein the single or double stranded RNA or the DNA virus is selected from the group consisting of retroviruses, poxviruses, immunodeficiency virus (HIV), echovirus, parvovirus, rubella virus, papillomaviruses, congenital rubella, Epstein-Barr virus, mumps, adenovirus, AIDS, chicken pox, cytomegalovirus, dengue, feline leukemia, fowl plague, hepatitis A, hepatitis B, HSV-1, HSV-2, hog cholera, influenza A, influenza B, Japanese encephalitis, measles, parainfluenza, rabies, respiratory syncytial virus, rotavirus, wart, yellow fever, adenovirus, a herpesvirus, a poxvirus, a picornavirus, an orthomyxovirus, a paramyxovirus, a coronavirus, a papovavirus, a hepadnavirus, a flavivirus, or a retrovirus.

31. A method for eliciting and/or enhancing B-cell and/or T-cell response against an antigen or a plurality of antigens, in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the anti-PD-1 immunocytokine of claim 13 in combination with the antigen or the plurality of antigens.

32. The method of claim 31 wherein the antigen or the plurality of antigen are conjugated to a DC-targeting antibody.

33. A pharmaceutical comprising the IL-15/IL-15 receptor alpha (IL-15Rα) fusion protein of claim 1 and a pharmaceutically acceptable carrier.

34. A pharmaceutical comprising the immunocytokine of claim 12 and a pharmaceutically acceptable carrier.

35. A vaccine composition comprising the immunocytokine of claim 13.

Description

FIGURES

[0121] FIG. 1: Anti-PD-1/IL-15/Rα immunocytokine designs that were profiled for functional activity and in vivo pharmacokinetic properties. Immunocytokines consist of (A) a knob-into-hole bi-specific design with one arm targeting PD-1 and the second delivering IL-15 and the IL-15 receptor a sushi domain (IC:K-in-H), (B) anti-PD-1 antibody with a C-terminal dockerin fusion that binds tightly to an IL-15/Rα fusion through an N-terminal cohesin domain (IC:Doc/Coh) and (C) an anti-PD-1 antibody with a C-terminal fusion of IL-15 and IL-15 Rα joined through flexible loops (IC:Fusion).

[0122] FIG. 2: Anti-PD-1/IL-15/Rα immunocytokines have enhanced recovery of exhausted CD8.sup.+ T cell proliferation relative to Keytruda. Recovery of proliferation in HIV-specific CD8.sup.+ T cells following stimulation with an HIV derived peptide. Results from multiple CFSE experiments are shown with enhance proliferation shown relative to Keytruda that was used as a positive control reference in each study.

[0123] FIG. 3: Comparison of IL-15/IL-15Rα immunocytokines directly fused to either a classical blocking (Keytruda) or a non-blocking (135C H1cL1c) anti-PD-1 antibody. Recovery of proliferation in HIV-specific CD8.sup.+ T cells following stimulation with an HIV derived peptide.

[0124] FIG. 4: Antigen-specific CD8 T cells proliferation observed in cells stimulated in the presence of Keytruda or Anti-PD-1/IL-15/Rα immunocytokines. PBMCs from a viremic HIV donor were stimulated with an HIV antigenic peptide and left in culture for 6-days before staining cells with a pentamer MHC-peptide complex.

[0125] FIG. 5: Pharmacokinetic profile of the anti-PD-1 antibody Keytruda in comparison with different anti-PD-1/IL-15/Rα immunocytokines. Total human IgG antibody was measured in the Keytruda samples and human IgG antibody with bound IL-15 was monitored for the immunocytokines.

[0126] FIG. 6. Experimental design. PBMCs from cART-patients at wk16 post vaccination (samples n=15) were stimulated with Gag-P24 peptides pools in the presence or not of, αPD1, IL-15/IL-15Rα, αPD1+IL-15/IL-15Rα or αPD1 fused to IL-15/IL-15Rα (fusion). After 44 hrs of stimulation cells were collected and stained then analyzed by flow cytometry to depict CD4.sup.+OX40.sup.+CD25.sup.+Foxp3.sup.+CD39.sup.+ specific-Tregs and CD4.sup.+OX40.sup.+CD25.sup.+Foxp3.sup.−CD39 specific effectors. Simultaneously, we assessed CD8.sup.+Pentamers.sup.+HIV-1 specific cell proliferation and cytokines production after a 6-day in vitro stimulation.

[0127] FIG. 7. CD4.sup.+HIV-1 specific responses. PBMCs from cART-patients at wk16 post vaccination (n=15) were stimulated with Gag P24 peptides pools in the presence or not of immuno-cytokines as explained in FIG. 2. After 44 hrs of stimulation cells were analyzed by flow cytometry to depict CD4.sup.+OX40.sup.+CD25.sup.+Foxp3.sup.+CD39.sup.+ specific-Tregs and CD4.sup.+OX40.sup.+CD25.sup.+Foxp3.sup.−CD39.sup.− specific-effectors.

[0128] FIG. 8. CD8.sup.+ specific responses. PBMCs from Dalia1 samples (n=5, but only 3 are shown) were CF SE-labeled and stimulated with Gag-P24 peptides pools in vitro and cultured in the presence (or not) of either αPD1 alone or α-PD1_IL-15/IL-15Rα fusion. Flow cytometry stainings show gating strategy for CD8.sup.+Pentamers.sup.+ specific cells (A), proliferation (B) and cytokines production (C).

[0129] FIG. 9. Experimental design for in vitro anti-CD40.HIV5pep-DC vaccine. PBMCs from cART patients (Physioph cohort, Hopital Henri-Mondor Creteil) were used. Samples (n=10) were used for CD14.sup.+ monocytes isolation (Miltenyi beads), differentiated, matured, activated and loaded with anti-CD40.HIV5pep (DC-vaccine) as performed for the Dalia1 trial (figurel). Experiments using PBMCs stimulated with Gag-P24 peptides pools or co-cultured with anti-CD40.HIV5pep in the presence (or not) of α-PD1 IL-15/IL-15Rα fusion during 44 hrs, have been performed. Gating strategies for CD4.sup.+OX40.sup.+CD25.sup.+ effectors and Tregs as applied above have been used.

[0130] FIG. 10. CD4.sup.+HIV-1 specific responses. Read-out experiments for FIG. 9 (above) showing Teff and Tregs specific cell frequencies. PBMCs stimulated with Gag-P24 peptides pools or co-cultured with anti-CD40.HIV5pep in the presence (or not) of α-PD1 IL-15/IL-15Rα fusion during 44 hrs, have been performed. Gating strategies for CD4.sup.+OX40.sup.+CD25.sup.+ effectors and Tregs as shown in FIG. 5 have been applied.

[0131] FIG. 11. CD4.sup.+ and CD8.sup.+HIV-1 specific responses. Read-out experiments for FIG. 9 (above) showing cytokines production from effector CD4.sup.+ and CD8.sup.+ T cells. 6 hrs before the end of the 44 hrs (OX40 assay), brefeldinA has been added in order to block intracellular cytokines secretion. Staining for IL-2/TNFα/IFNγ has been performed and cells were analyzed by flow cytometry. Blue histograms show increased cytokine production when α-PD1 IL-15/IL-15Rα fusion was added to PBMCs/anti-CD40.HIV5pep-DC cocultures.

[0132] FIG. 12. Immunotherapy induced suppression of tumor growth in the PD-1 HuGEMM in vivo Panc02 tumor model. PD-1 huGEMM mice were implanted with Panc02 tumor cells and upon reaching a mean tumor volume of 100 mm.sup.3, were treated twice per week with PBS or one of the four immunotherapies indicated. The 10 mice allocated to each group were monitored for tumor volume twice per week with the mean volume show versus days under therapy.

[0133] FIG. 13: The NB01/IL-15/IL-15Rα immunocytokine exert a significant suppression in Panc02 tumor growth from Day 7 to 24 following immunotherapy initiation. The anti-tumor effect of the different therapies was evaluated by calculating the area under the curve (AUC) from Day 7 to Day 24 following initiation of the study. Statistical differences between the PBS control and the individual therapies was determined using the non-parametric Mann-Whitney test. Analysis was performed using Graphpad Prism 8.3.0.

[0134] FIG. 14: NB01/IL-15/IL-15Rα immunocytokine therapy prolongs the survival of Panc02 implanted PD-1 HuGEMM mice. The percentage of mouse survival over time following initiation of therapy in Panc02 implanted mice was evaluated by Kaplan-Meier survival curve analysis. The log rank test confirmed that both the NB01/IL-15/IL-15Rα immunocytokine monotherapy and the Keytruda+IL-15/IL-15Rα ALT-803 super agonist dual therapy significantly increased the time of mouse survival. In contrast, neither Keytruda monotherapy alone nor the Keytruda/IL-15/IL-15Rα immunocytokine monotherapy significantly prolonged survival of the Panc02 implanted mice. Analysis was performed using Graphpad Prism 8.3.0.

EXAMPLE 1

[0135] In the design of immunocytokines, our focus was on identifying therapeutic agents that had improved potency in enhancing antigen specific T cell proliferation relative to anti-PD-1 therapy and a sufficiently long in vivo half-life. A second consideration was the production yield and biophysical stability of these immunocytokines that will facilitate the advancement of a promising candidate for in vivo testing and pre-clinical profiling. Three separate immunocytokine designs were evaluated as illustrated in FIG. 1. The immune-enhancing activity of the novel anti-PD-1/IL-15/Rα was evaluated in vitro in a highly standardized CF SE proliferation assay using blood mononuclear cells from chronically infected HIV infected donors. HIV-specific T cells from these donors have been exposed to high levels of antigen over an extended period of time and as such are functionally exhausted, express high levels of the PD-1 immune checkpoint inhibitors and have a poor proliferative response in the presence of antigen-specific stimulation. Stimulation of the blood mononuclear cells with HIV derived peptides followed by six days in culture led to an increase in CFSE low CD8 T cells that have undergone proliferation. Addition of the classical PDL-1 blocking anti-PD-1 Ab Keytruda led to an enhanced level of proliferation relative to the peptide alone control thus indicating that anti-PD-1 Abs recover CD8.sup.+ T cells from exhaustion (FIG. 2). The three different immunocytokine constructs tested in parallel gave a minimum of a 2 to 4-fold increased level of CD8.sup.+ T cell proliferation relative to the Keytruda control anti-PD-1 antibody. Importantly, the IC:Doc/Coh and IC:Fusion constructs had significantly improved immune-enhancing effects relative to Keytruda even at 100-fold lower protein concentrations. The IC:K-in-H construct induced increased levels of CD8 T cell proliferation relative to Keytruda at 1 μg/ml, however IC:K-in-H was >10-fold less potent than IC:Doc/Coh and IC:Fusion constructs. In a separate experiment, an additional immunocytokine was generated using the IC:Fusion construct design in combination with the 135C H1cL1c anti-PD-1 antibody that is non-blocking of the PD-1/PDL-1 interaction (Fenwick et al, 2019). The functional activity of 135c directly fused to IL-15/IL-15Rα in the CFSE proliferation assay was found to be comparable to the IC:Fusion that is a Keytruda-IL-15/IL-15Rα fusion (FIG. 3). Interestingly, with the blood mononuclear cells from this viremic HIV infected donors, the two different immunocytokines at 0.1 μg/ml induced up to a 20-fold increase in CD8 T cell proliferation relative to Keytruda at 5 μg/ml.

EXAMPLE 2

[0136] The increased level of CD8.sup.+ T cell proliferation in the presence of the different immunocytokine highlights their superior functional activity relative to anti-PD-1 therapy alone. However, equally important is that CD8.sup.+ T cell expansion is specific for the HIV peptide antigen used in the stimulation. The specificity of the enhanced CD8.sup.+ T cell proliferation in the presence of immunocytokines was determined by staining of CFSE low proliferating CD8.sup.+ T cells with the pentamer MHC-HIV peptide complex. Flow cytometry results in FIG. 4 show that with Keytruda, IC:K-in-H, IC:Doc/Coh and IC:Fusion, the majority (60-80%) of the proliferating CD8.sup.+ T cells are MHC pentamer positive and specific for the HIV peptide antigen used in the stimulation. In contrast, stimulations with the SEB T cell superantigen results in only 2% of the proliferating CD8.sup.+ T cells staining positive for the MHC-HIV peptide complex. These pentamer staining studies confirm that the immunocytokine complexes induce a strong and specific induction CD8.sup.+ T cells proliferation in the presence of antigenic stimulation.

EXAMPLE 3

[0137] To further characterize these immunocytokines as therapeutic agents, pharmacokinetic studies were performed in C57BL/6 mice that were dosed with the 2 mg/kg of the different drugs and serum samples were collected over the following 7 days. PK properties of Keytruda and the three immunocytokine were determined using Luminex assays to detect human IgG or human IgG with bound IL-15 over the course of the study (FIG. 5). The IC:K-in-H construct, with a more native antibody structure relative to the other immunocytokines, had an in vivo half-life that was closest to the Keytruda antibody (tin, —5 days). However, the drug exposure was also 1 log lower than Keytruda with equivalent doses administered to the mice. Given that the IC:K-in-H construct is only about 10-fold more potent than Keytruda, the inferior PK is likely to negate any functional advantages offered by this immunocytokine. The IC:Doc/Coh and IC:Fusion immunocytokines have a terminal half-lives of approximately 1 day and are more rapidly cleared from mice compared to Keytruda. However, this half-life is significantly longer that the <1 hr half-life reported for IL-15 in vivo and drug levels of these two immunocytokines at day 7 are still be high (>0.01 μg/ml) relative to the significantly improved in vitro functional effect of IC:Doc/Coh and IC:Fusion compared to Keytruda. In contrasting the different properties of the three immunocytokines evaluated, the IC:Fusion has the best overall profile with: 1) a consistent >2-fold improved functional activity relative to Keytruda over a 2 log range in concentration, 2) antigen specific enhanced proliferation of CD8.sup.+ T cells as demonstrated by MHC petamer staining, and 3) PK profile that would support a once weekly dosing regimen. An additional advantage of the IC:Fusion construct compared to IC:Doc/Coh, is that it lacks the dockerin and cohesin domains that may be prone to increased immunogenic and would be detrimental to the PK profile in animals that were treated with repeat dosing.

EXAMPLE 4

[0138] Based on our previous results, we reasoned that therapeutic DC-based vaccination could be combined with an immuno-modulatory cytokine fused to an immune checkpoint inhibitor in order to increase T cell responses. To this end, we used an αPD-1 monoclonal antibody (Keytruda, a clinical molecule) that proved its' potency in the cancer field and IL-15/IL-15Rα, a cytokine known to impact effector CD8.sup.+ T cell proliferation.

[0139] PBMCs from weekly post-vaccination (Dalia1, n=15) were stimulated in vitro with Gag-P24 peptides pools in the presence or not of αPD-1_IL-15/IL-15Rα fusion, αPD1 alone, IL-15/IL-15α alone or αPD-1+IL-15/IL-15α (refer to experimental design, FIG. 6). After 44 hrs stimulation, CD4.sup.+OX40.sup.+CD25.sup.+HIV-1 specific cells have been analyzed.

[0140] We demonstrate that in the presence of α-PD-1_IL-15/IL-15Rα fusion, Gag-P24 stimulated PBMCs from cART HIV-1.sup.+ vaccinated patients led to a significant increase in HIV-1 specific T cell responses as compared to the other conditions. In FIG. 7, panel A, we show that CD4.sup.+OX40.sup.+CD25.sup.+HIV-1 specific cell frequency, that includes both effectors and regulatory cells (Teff and Tregs), increased significantly when α-PD-1_IL-15/IL-15Rα fusion was added in Gag P24- stimulated PBMCs. Addition of αPD-1 did not increase the frequency CD4.sup.+ specific cells as compared to Gag P24 stimulated cells alone, while IL-15/IL-15Rα or IL-15/IL-15Rα+αPD-1 addition was slightly better but did not reach the potency obtained with α-PD-1_IL-15/IL-15Rα fusion. In FIG. 7 panel B, we show similar trends when zooming on effectors (OX40.sup.+CD25.sup.+CD39.sup.−Foxp3.sup.−), and interestingly we observed a significant decrease in Tregs (OX40.sup.+CD25.sup.+CD39.sup.+Foxp3.sup.+) when α-PD-1_IL-15/IL-15Rα fusion was added. These data demonstrate that fusion of αPD-1 with IL-15/IL-15Rα is a good way to boost HIV-1 specific effector CD4.sup.+ T cells in vitro.

EXAMPLE 5

[0141] In order to depict CD8.sup.+HIV-1 specific cells we used pentamers staining (Proimmune, UK) and flow cytometry analyses after in vitro proliferation of PBMCs from 6 patients (Dalia1, week16) that were stimulated with HLA-restricted peptides pools. After a 6 day-in vitro proliferation we performed an overnight re-stimulation with the specific peptides and performed CFSE stainings and flow cytometry to measure CD8.sup.+ proliferation and intracellular cytokine production (IL-2, TNFα, IFN-γ). The results demonstrated that PBMCs stimulation in the presence of α-PD-1_IL-15/IL-15Rα fusion significantly increased CD8.sup.+ pentamers.sup.+ cell proliferation and cytokines production, as compared to αPD1 alone, which showed similar responses when compared to the condition with only peptides stimulation (FIG. 8). The graphs show the results obtained with 3 patients out of 5.

EXAMPLE 6

[0142] In order to demonstrate that αPD-1_IL-15/IL-15Rα fusion could be also used with another DC-based vaccine (anti-CD40.HIV5pep-DC), we performed in vitro experiments where anti-CD40.HIV5pep construct has been loaded on matured and differentiated CD14.sup.+ monocytes as shown in the experimental design below (FIG. 9 and Cobb et al. JIM 2011), before co-cultures with autologous PBMCs. We used the OX40 assay to depict CD4.sup.+ specific T cells by flow cytometry as shown in the lower panel of FIG. 9.

[0143] Of note, for the read-out experiments using the OX40 assay, we performed 2 conditions: in the first one we stimulated PBMCs from patients with Gag P24 in the presence or not of αPD-1_IL-15/IL-15Rα fusion and in the second condition we cocultured PBMCs with anti-CD40.HIV5pep-DC in the presence or not of αPD-1_IL-15/IL-15Rα fusion. The results shown in FIG. 10, demonstrated that addition of αPD-1_IL-15/IL-15Rα fusion in both conditions enhanced CD4.sup.+HIV-1 specific effector cells (blue histograms), but this increase was more significant when PBMCs were stimulated/co-cultured with anti-CD40.HIV5pep-DC. Moreover, we observed a significant decrease in CD4.sup.+HIV-1 specific Tregs (histogram, lower panel).

[0144] Finally, we measured intracellular cytokines (IL-2/TNFα/IFNγ) in both CD4+ and CD8.sup.+ cells (addition of brefeldin A 6 hrs before the end of the 44 hrs of OX40assay). FIG. 11 shows a significant increase in cytokines production when PBMCs were co-cultured with anti-CD40.HIV5pep_DCs in the presence of αPD-1_IL-15/IL-15Rα fusion.

[0145] Altogether these results demonstrate that i) DC-targeting (PBMCs co-cultured with anti-CD40.HIV5pep-DCs) leads to better T cell responses as compared to Gag P24-stimulated PBMCs and ii) combination of αPD-1_IL-15/IL-15Rα fusion boosted CD8.sup.+-specific responses (proliferation and cytokines production) and decreased CD4.sup.+-specific Tregs, suggesting that αPD-1_IL-15/IL-15Rα fusion is good tool that can be pushed to the clinic.

EXAMPLE 7: Efficacy of Anti-PD-1/IL-15/IL15Rα Immunocytokines in the in Vivo Panc02 Mouse Tumor Model

[0146] The objective of this study was to evaluate the in vivo therapeutic efficacy of the anti-PD-1/IL-15/IL-15Rα immunocytokine test agents described in this application in the treatment of a subcutaneous Panc02 murine pancreatic cancer xenograft in female HuGEMM hPD-1 mice. The Panc02 tumor cells are poorly immunogenic and represent a challenging tumor model for most cancer immunotherapies.

[0147] The HuGEMM PD-1 model performed by CrownBio was developed by knocking-in human exon 2 to replace its mouse PD-1 counterpart. This allows for the in vivo efficacy evaluation of human therapeutic antibodies, which recognize the humanized PD-1 receptor. Mice of age 6-8 weeks were inoculated with 3×10.sup.6 Panc02 tumor cells in 0.1 mL of PBS and the study was initiated 7 days later when the mean tumor size reaches approximately 100 (70-130) mm.sup.3. All animals were randomly allocated to five study group arms based on “Matched distribution” method (StudyDirector™ software, version 3.1.399.19) with 10 mice per group. The five investigational arms of the study included: 1) PBS untreated control, 2) pembrolizumab (Keytruda®) anti-PD-1 twice weekly treatment at 5 mg/kg, 3) pembrolizumab (Keytruda®) at 5 mg/kg+0.1 mg/kg of the IL-15/IL-15Rα ALT-803 super agonist, both administered twice weekly, 4) Keytruda fused to IL-15/IL-15Rα immunocytokine (IC) administered at 2 mg/kg twice weekly and 5) NB01b anti-PD-1 antibody fused to IL-15/IL-15Rα immunocytokine (IC) administered twice weekly at 2 mg/kg. Keytruda used in the study was a clinical lot of antibody purchased from the Lausanne University Hospital. The IL-15/IL-15Rα ALT-803 super agonist and immunocytokines were recombinant proteins produced independently through transient transfection of CHO express or HEK 293T mammalian cell lines. The proteins were expressed with signal sequences that was cleaved upon secretion from the transfected cells. Each of the therapeutic proteins was then purified from the cell medium using a protein A affinity column. Following buffer exchange through dialysis against PBS, therapeutic agents were verified with an limulus amebocyte lysate (LAL) kit from Charles River and endotoxin levels determined to be less than 1 EU/ml. All therapeutic agent were administered intraperitoneal as a solution in PBS buffer. Tumor volumes in all mice was measured twice per week in two dimensions using a caliper, and the volume expressed in mm.sup.3 using the formula: volume=(length×width×width)/2. A tumor volume cutoff of 1500 mm.sup.3 was selected for establishing mouse survival criteria in this study.

[0148] Longitudinal evaluation of mouse tumor volumes in each of the study arms showed that at Day 10 under therapy, all anti-PD-1 based therapies showed signs of tumor suppression relative to the PBS untreated control mice (FIG. 12). The Keytruda alone and Keytruda/IL-15/IL-15Rα IC therapies exerted only transient suppression of tumor growth with increases in mean volume observed at Day 14 of the study. In contrast, Keytruda+ALT-803 super agonist and the NB01/IL-15/IL-15Rα IC therapies demonstrated a prolonged in vivo functional activity with an increase in mean tumor volume observed only seven days later on Day 21 of the study. After this point, the Panc02 cells escaped suppression by the different immunotherapies and tumor growth appeared to progress at a similar rates in all anti-PD-1 based therapeutic arms.

[0149] The relative levels of tumor suppression between the different therapies and the PBS arm was evaluated by comparing the tumor volume area under the curve values for Days 7 to 24 of the study (FIG. 13). The strongest tumor suppression over this period was observed in the Keytruda+super agonist and NB01/IL-15/IL-15Rα IC study arms. Relative to the PBS control mice, we observed a significant tumor suppression in Keytruda+super agonist (p=0.0021) and NB01/IL-15/IL-15Rα IC (p=0.0041) therapeutic arms with a less significant reduction in tumor volume for the Keytruda alone therapy (p=0.018).

[0150] The anti-tumor efficacy of the different therapies was also evaluate using a Kaplan-Meier Survival Curve analysis (FIG. 14). The NB01/IL-15/IL-15Rα immunocytokine monotherapy exerted a statistically significant 14 day increase in mouse survival relative to the PBS control (Log rank test; p=0.029) that was slightly longer than the 12 day increased survival in the dual combination therapy of Keytruda+IL15/IL15Rα super agonist dual therapy (Log rank test; p=0.049). In contrast, both Keytruda and Keytruda/IL-15/IL-15Rα immunocytokine monotherapies did not significantly increase the survival time of mice implanted with the poorly immunogenic Panc02 tumor cells.

[0151] Overall, these studies show that the immunocytokine fusion of the NB01b anti-PD-1 antibody with the IL-15 and IL-15Rα has a significant functional activity in both suppressing Panc02 tumor grown and in prolonging mouse survival in comparison to the untreated mice. This demonstrated activity of the immunocytokine was equivalent in efficacy as compared to the Keytruda+super agonist dual therapy.

REFERENCES

[0152] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.